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Encapsulation of amoxicillin within laponite-doped poly(lactic-<italic>co</italic>-glycolic acid) nanofibers: preparation, characterization, and antibacterial activity
Encapsulation of Amoxicillin within Laponite-Doped Poly(lactic-co-glycolic acid) Nanoﬁbers: Preparation, Characterization, and Antibacterial ActivityShige Wang,Fuyin Zheng,Yunpeng Huang,Yuting Fang,Mingwu Shen,Meifang Zhu,and Xiangyang Shi†State Key Laboratory for Modiﬁcation of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, People's Republic of China ‡College of Materials Science and Engineering, Donghua University, Shanghai 201620, People's Republic of China §College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People's Republic of China ⊥CQM-Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal ABSTRACT: We report a facile approach to encapsulatingamoxicillin (AMX) within laponite (LAP)-doped poly(lactic-co-glycolic acid) (PLGA) nanoﬁbers for biomedical applica-tions. In this study, a synthetic clay material, LAP nanodisks,was ﬁrst used to encapsulate AMX. Then, the AMX-loadedLAP nanodisks with an optimized AMX loading eﬃciency of9.76 ± 0.57% were incorporated within PLGA nanoﬁbersthrough electrospinning to form hybrid PLGA/LAP/AMXnanoﬁbers. The loading of AMX within LAP nanodisks and theloading of LAP/AMX within PLGA nanoﬁbers werecharacterized via diﬀerent techniques. In vitro drug release proﬁle, antimicrobial activity, and cytocompatibility of the formedhybrid PLGA/LAP/AMX nanoﬁbers were also investigated. We show that the loading of AMX within LAP nanodisks does notlead to the change of LAP morphology and crystalline structure and the incorporation of LAP/AMX nanodisks does notsigniﬁcantly change the morphology of the PLGA nanoﬁbers. Importantly, the loading of AMX within LAP-doped PLGAnanoﬁbers enables a sustained release of AMX, much slower than that within a single carrier of LAP nanodisks or PLGAnanoﬁbers. Further antimicrobial activity and cytocompatibility assays demonstrate that the antimicrobial activity of AMX towardthe growth inhibition of a model bacterium of Staphylococcus aureus is not compromised after being loaded into the hybridnanoﬁbers, and the PLGA/LAP/AMX nanoﬁbers display good cytocompatibility, similar to pure PLGA nanoﬁbers. With thesustained release proﬁle and the reserved drug activity, the organic/inorganic hybrid nanoﬁber-based drug delivery system may ﬁnd various applications in tissue engineering and pharmaceutical science.
KEYWORDS: poly(lactic-co-glycolic acid), laponite, electrospinning, amoxicillin, sustained release, antimicrobial activity method, drug molecules are directly integrated within the The distinctive features of nanoﬁbers such as ﬂexibility in nanoﬁbers by simply electrospinning the drug/polymer mixture surface functionalities, superior mechanical durability, and solution or by absorbing/assembling the drugs onto the interconnected and readily controlled secondary structures nanoﬁber surfaces or in the interior of nanoﬁbers− aﬀord them to be used as a unique drug delivery system, which Although this method allows easy incorporation of drug has inherent advantages including easy implementation, little molecules within the nanoﬁbers, a burst release often occurs, inﬂuence on the drug activity, and well controlled drug release which is not desirable in most cases.Emulsion and coaxial rate.−Electrospinning is a simple and straightforward way to electrospinning are two improved techniques to be used for produce nanoﬁbers with designed structure and morphol- drug delivery applications, which is able to mitigate the burst ogy.Since Kenawy et al. ﬁrst examined the drug release release of the drug to some extent.In both methods, the property from electrospun nanoﬁbers, the use of electrospun drugs are able to be embedded into the core region of the nanoﬁbers for drug delivery applications has received increasing nanoﬁbers in a reservoir-type model and form a so-called interest in the pharmaceutical ﬁUntil now, a number ofdiﬀerent drug-loading methods have been developed via September 27, 2012 conventional, emulsion, or coaxial electrospinning techni- Accepted: November 6, 2012 ques.−In the conventional single ﬂuid electrospinning Published: November 6, 2012 2012 American Chemical Society dx.doi.org/10.1021/am302130b ACS Appl. Mater. Interfaces 2012, 4, 6393−6401
ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of the Loading of Free AMX Drug within PLGA Nanoﬁbers (a) and the Loading of AMX/LAPwithin PLGA Nanoﬁbers (b) "core−sheath" structure. In this reservoir-type structure, the Staphylococcus aureus (S. aureus) as a model bacterium both in outer polymer shell can act as an additional barrier to control liquid and on solid medium. Finally, the cytocompatibility of the drug release proﬁNevertheless, there are still some PLGA/LAP/AMX nanoﬁbers was evaluated through 3-(4,5- issues existing in the emulsion and coaxial electrospinning techniques. For example, the coaxial electrospinning may need colorimetric assay and scanning electron microscopy (SEM) substantial optimization of the electrospinning parameters, and morphology observation of porcine iliac artery endothelial cells the emulsiﬁer used in emulsion electrospinning may cause (PIEC) cultured onto the nanoﬁber scaﬀold. To our knowl- compromised biocompatibility of the nanoﬁbers.Therefore, edge, this is the ﬁrst report related to the development of development of other nanoﬁber systems that can overcome the PLGA/LAP composite nanoﬁbers for drug delivery applica- burst release of the encapsulated drugs still remains a great In our previous study, we reported the use of halloysite ■ EXPERIMENTALSECTION nanotubes (HNTs)/poly(lactic-co-glycolic acid) (PLGA) com- Materials. PLGA (Mw = 81 000 g/mol) with a lactic acid/glycolic posite nanoﬁbers for encapsulation and release of a model drug acid ratio of 50:50 and LAP were purchased from Jinan Daigang tetracycline hydrochloride In this approach, the TCH Biotechnology Co., Ltd. (China) and Zhejiang Institute of Geologic drug molecules were ﬁrst physically encapsulated within the and Mineral Resources (China), respectively. AMX was from Shanghai HNTs, followed by electrospinning the mixture solution of Yuanye Biotechnology Co., Ltd. S. aureus was purchased from PLGA and TCH-loaded HNTs to form a composite drug- Shanghai Fuzhong Biotechnology Development Co., Ltd. Luria- incorporated nanoﬁber, which was proven to be able to Bertani (LB)-medium and agar were from Beijing AoboxingBiotechnology Co., Ltd., tetrahydrofuran (THF), and N,N-dimethyl- signiﬁcantly alleviate the burst release of the TCH. This formamide (DMF) were from Sinopharm Chemical Reagent Co., Ltd.
preliminary success leads us to hypothesize that other naturally (China). PIEC cells were obtained from Institute of Biochemistry and occurring or synthetic clay materials that have been used for Cell Biology (the Chinese Academy of Sciences, Shanghai).
drug delivery applications may also be able to be incorporated Dulbecco's Modiﬁed Eagle's medium (DMEM), fetal bovine serum within polymer nanoﬁbers to improve the drug release proﬁle (FBS), penicillin, and streptomycin were purchased from Hangzhou for various biomedical applications. As a synthetic clay material, Jinuo Biomedical Technology (Hangzhou, China). All chemicals and laponite (LAP) has been used as a drug carrier because the reagents were used as received. Water used in all experiments was interlayer space of LAP can be used for eﬀective drug puriﬁed using a Milli-Q Plus 185 water puriﬁcation system (Millipore, encapsulation with high retention capacity.−For example, Bedford, MA) with resistivity higher than 18 MΩ·cm.
Preparation of Drug-Loaded LAP Nanodisks. AMX was ﬁrst Jung et al. reported the incorporation of a hydrophobic drug dissolved into water to obtain AMX aqueous solutions with diﬀerent itraconazole (ITA) into LAP through an interfacial interaction concentrations (0.1, 0.3, 0.5, 1, and 2 mg/mL, respectively) at room of LAP and ITA. However, their release data showed that the temperature. Then, LAP nanodisks were dispersed into the resulting release of ITA from ITA/laponite hybrid could reach 75% AMX solutions with diﬀerent concentrations (3, 5, and 10 mg/mL, during the ﬁrst 24 h.Take the excellent biodegradability, respectively) to get a batch of LAP/AMX suspensions. After that, the biocompatibility, and electrospinnability of PLGA into LAP/AMX suspensions were magnetically stirred for 24 h in order to account,it is expected that PLGA/LAP composite make the LAP fully swell and to make the AMX molecules be nanoﬁbers may be used as drug carriers to aﬀord the suﬃciently intercalated into the interlayer of LAP. The LAP/AMXnanodisks were then separated by centrifugation (8000 rpm, 5 min) encapsulated drugs with a sustained release proﬁle.
and washed with water for 3 times to remove the excessive AMX. The In this present study, LAP nanodisks were ﬁrst used to supernatants after 4 times centrifugation were collected together, and encapsulate a model drug of amoxicillin (AMX). Then, the the nonencapsulated AMX was quantiﬁed using a Lambda 25 UV−vis LAP/AMX nanodisks were incorporated within PLGA nano- spectrophotometer (Perkin-Elmer, USA) at 230 nm with a ﬁbers via electrospinning to form PLGA/LAP/AMX nanoﬁbers concentration−absorbance calibration curve at the same wavelength.
(Scheme ). The formed LAP/AMX nanodisks and the Finally, the LAP/AMX nanodisks were obtained by lyophilization. The composite PLGA/LAP/AMX nanoﬁbers were intensively drug loading eﬃciency can be calculated from the following equation: characterized using diﬀerent techniques. In vitro drug release loading efficiency = M /M × 100% behavior of the composite PLGA/LAP/AMX nanoﬁbers was examined using UV−vis spectroscopy. The antimicrobial where Mt and M0 stand for the mass of encapsulated AMX and the activity of the composite nanoﬁbers was investigated using initial total AMX used for encapsulation, respectively.
dx.doi.org/10.1021/am302130b ACS Appl. Mater. Interfaces 2012, 4, 6393−6401 ACS Applied Materials & Interfaces Preparation of AMX-Loaded Electrospun Nanoﬁbers. PLGA fresh PBS solution was replenished. The optical density (OD) value was dissolved in a mixed solvent of THF/DMF (v/v = 3:1) with an was measured using a Lambda 25 UV−vis spectrophotometer at 230 optimized concentration of 25% (w/v).After that, AMX (0.5 wt % relative to PLGA) or LAP/AMX (with ﬁnal 0.5% AMX relative to In Vitro Antibacterial Activity Assay. The antibacterial activity PLGA) was dispersed into PLGA solution for subsequent electro- of LAP/AMX nanodisks, PLGA/AMX nanoﬁbers, and PLGA/LAP/ spinning to form PLGA/AMX or PLGA/LAP/AMX nanoﬁbers, AMX nanoﬁbers was evaluated in liquid medium (2.5 g LB medium respectively (Scheme PLGA/LAP nanoﬁbers without AMX but dissolved into 100 mL water) by recording the absorbance of the with the same amount of LAP used to encapsulate 0.5% AMX (relative solution at 625 nm using a Lambda 25 UV−vis spectrophotometer, to PLGA) were also prepared as a control material. The electro- which is in direct proportion to the bacterial number.In brief, 5 spinning system was made up of a syringe pump with a 10 mL syringe, mL of the bacterial solution with an OD value of 0.1−0.2 at 625 nm a silicone hose, a stainless steel needle with an inner diameter of 0.8 was added into each 15 mL glass tube. Then, AMX powder, LAP/ mm, a high voltage power supply, and a thin aluminum foil acting as a AMX nanodisks, PLGA/AMX nanoﬁbers, and PLGA/LAP/AMX collector which was positioned horizontally and grounded. A clamp nanoﬁbers were added into each tube with the AMX concentration of was used to connect the high voltage power supply with the needle.
10, 20, and 30 μg/mL, respectively. AMX powder was used as a The electrospinning process was carried out under ambient condition positive control, while PLGA and PLGA/LAP nanoﬁbers without with a ﬁxed electrical potential of 20 kV, a collect distance of 15 cm, AMX were used as negative controls. Tube without sample was set as and a feeding rate of 0.8 mL/h by a syringe pumpAfter another negative control. All the samples were in triplicate and electrospinning, nanoﬁbers were taken oﬀ from the collector and incubated at 37 °C with a shaking speed of 100 rpm for 24 h. After vacuum-dried for at least 48 h to remove the residual organic solvent that, the OD value at 625 nm was monitored using UV−vis and moisture.
spectroscopy. The bacterial inhibition percentage can be calculated Characterization Techniques. The LAP and LAP/AMX nano- by the following eqution: disks were characterized using Fourier transform infrared (FTIR)spectroscopy. The analysis was performed using a Nicolet Nexus 670 bacterial inhibition (%) = I ( − I )/I × 100 FTIR (Nicolet-Thermo) spectrometer. All spectra were recorded using where Ic and Is are the average ODs of the control group and the a transmission mode with a wavenumber range of 650−4000 cm−1.
experimental group, respectively. The above method was also used to The morphology of LAP and LAP/AMX nanodisks was observed evaluate the correlation between the antibacterial activity of the using ﬁeld emission scanning electron microscopy (FESEM) PLGA/LAP/AMX nanoﬁbers as a function of the release time. Brieﬂy, (HITACHI S-4800, Japan) with an accelerating voltage of 15 kV.
PLGA/LAP/AMX nanoﬁbers with the AMX mass of 100 μg were The LAP or LAP/AMX nanodisks were ﬁrst dispersed into water.
added to a glass tube containing 5 mL of the bacterial suspension with Then, the suspension of LAP or LAP/AMX nanodisks was dropped an OD value of 0.1−0.2 at 625 nm. The bacterial inhibition percentage onto an aluminum foil, air-dried, and sputter-coated with a carbon ﬁlm was determined at diﬀerent time points (1, 2, 6, 12, 24, 48, 72, and 96 with a thickness of 10 nm before measurement. The crystalline h, respectively). For comparison, PLGA/AMX nanoﬁbers were also structure of LAP, AMX, and LAP/AMX nanodisks was characterized tested under similar conditions.
by a Rigaku D/max-2550 PC X-ray diﬀraction (XRD) system (Rigaku Another antibacterial activity testing method based on solid Co., Tokyo, Japan) using Cu Kα radiation with a wavelength of 1.54 Å medium was also used in this study.Brieﬂy, agar (1.5 g) was at 40 kV and 200 mA. The scan was performed from 5° to 60° (2θ).
added into 100 mL liquid medium and autoclaved. Then, the agar The plane spacing of diﬀerent diﬀraction planes (dhkl) can be medium was poured onto Petri dishes and air-dried. The PLGA, calculated from the Bragg's Law: PLGA/LAP, PLGA/AMX, and PLGA/LAP/AMX nanoﬁbrous mats were cut into small pieces with a diameter of about 1 cm and the same weight. After that, the solid agar medium plates were seeded with 100 μL of S. aureus suspension and covered with PLGA, PLGA/LAP, where λ is the wavelength of the copper anode source (λ = 1.54 Å) and PLGA/AMX, and PLGA/LAP/AMX nanoﬁbrous mats, respectively, θ stands for the diﬀraction angle of each indexed diﬀraction plane. The for an antibacterial activity assay. In another method, the solid agar morphology of PLGA, PLGA/LAP, PLGA/AMX, and PLGA/LAP/ medium plates were ﬁrst covered with the PLGA, PLGA/LAP, PLGA/ AMX nanoﬁbers was observed using scanning electron microscopy AMX, and PLGA/LAP/AMX nanoﬁbrous mats, respectively, and the (SEM) (JEOL JSM-5600LV, Japan) with an accelerating voltage of 10 ﬁbrous mats were removed after a 4 h incubation. Then, 100 μL S.
kV. Before measurement, each sample was sputter-coated with a 10 aureus suspension was seeded onto each sample-treated solid medium.
nm-thick gold ﬁlm. Fiber diameter was measured using Image J 1.40 G All of these agar plates were incubated at 37 °C for the given time software (At least 100 period. The bacterial inhibition zones were visually observed to test nanoﬁbers from diﬀerent SEM images for each sample were randomly the samples' antibacterial activity.
selected and analyzed. Water contact angle test was used to evaluate Cytocompatibility Evaluation. For cytocompatibility evaluation, the surface hydrophilicity of the PLGA/AMX and PLGA/LAP/AMX PLGA and PLGA/LAP/AMX nanoﬁbers were prepared on coverslips ﬁbrous mats as reported in our previous study.In brief, a pendant with a diameter of 14 mm. Then, these mats were ﬁxed in 24-well droplet of water with 1 μL drop size was dropped onto the surface of plates with stainless steel rings and sterilized with 75% alcohol for 2 h.
each sample at the randomly selected area at ambient temperature and After that, all wells with samples were washed 3 times with PBS humidity. The contact angle was measured three times for each sample solution to remove the residual alcohol. Finally, 1 mL of complete using a contact angle goniometer (DSA-30, Kruss, Germany) when the DMEM was added to individual wells to incubate at 37 °C overnight.
droplet was stable.
PIEC cells were seeded at a density of 1.5 × 104 cells/well for MTT In Vitro Drug Release. The in vitro release kinetics of AMX from assay and 2 × 104 cells/well for SEM morphology observation, LAP/AMX nanodisks, PLGA/AMX nanoﬁbers, and PLGA/AMX/ respectively. Coverslips without nanoﬁbers and tissue culture plates LAP nanoﬁbers was studied using UV−vis spectroscopy. Brieﬂy, LAP/ (TCPs) were used as controls.
AMX nanodisks (6 mg) were dispersed into 1 mL of phosphate After cell seeding for 8 h or 3 days, unattached cells were washed buﬀered saline (PBS) solution (pH = 7.4) and placed in a dialysis bag out with PBS solution and MTT solution (40 μL) diluted with fresh with a molecular weight cutoﬀ of 10 000 and then dialyzed against 2 medium (360 μL) was added to each well. After being incubated at 37 mL of PBS solution in a sample vial. For the nanoﬁbers, 24 mg of °C for 4 h, 400 μL of DMSO was added to dissolve the purple MTT PLGA/AMX or PLGA/AMX/LAP nanoﬁbers was dipped into a formazan crystal. Then, 100 μL of the dissolved formazan solution of sample vial containing 3 mL of PBS solution. All these samples were in each sample was transferred into individual wells of a 96-well plate to triplicate and were incubated in a vapor-bathing constant temperature test the OD value at 570 nm using a microplate reader (MK3, vibrator at 37 °C for diﬀerent time periods. At each time interval, 1 mL Thermo, USA). Mean and standard deviation for the triplicate wells of PBS solution was taken out from each vial and an equal volume of for each sample were reported.
dx.doi.org/10.1021/am302130b ACS Appl. Mater. Interfaces 2012, 4, 6393−6401
ACS Applied Materials & Interfaces After being cultured for 8 h or 3 days, samples were rinsed 3 times aggregation of the LAP nanodisks at higher concentrations, with PBS solution and then ﬁxed with 2.5 wt % glutaraldehyde at 4 °C leading to decreased accessibility of the drug molecules to the for 2 h. After that, the samples were dehydrated through a series of interlayer space of LAP. We also note that the optimized gradient ethanol solutions of 30%, 50%, 70%, 80%, 90%, 95%, and loading eﬃciency of 9.76 ± 0.57% may not be the highest 100% and air-dried overnight. The morphology of cells was observed loading eﬃciency; further adjusting the concentrations of LAP, by SEM (JEOL JSM-5600LV) with an accelerating voltage of 10 kV,and the samples were sputter coated with a 10 nm thick gold ﬁlm AMX, and the solution pH is necessary to achieve the maximum loading eﬃciency.
Statistical Analysis. One-way ANOVA statistical analysis was The successful encapsulation of AMX within LAP was performed to compare the cytocompatibility of cells cultured onto conﬁrmed using FTIR spectroscopy (Figure In the FTIR diﬀerent materials and to compare the bacterial inhibition eﬀect of the spectrum of AMX powder (Figure the typical absorption tested materials with diﬀerent AMX concentrations in liquid medium.
bands at 1687, 1519, and 1235 cm−1 can be assigned to the 0.05 was selected as the signiﬁcance level, and the data were indicated amide I, amide II, and amide III bond of AMX, respectively.
with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, The weak peaks at 1770 and 1397 cm−1 may be attributed to the vibration of carbonyl group and carboxyl group of the RESULTS AND DISCUSSION AMX, respectively.The peaks at 3180 and 3050 cm−1 areassigned to the stretching vibration of free amino group in the Loading of AMX within LAP Nanodisks. Diﬀerent from AMX structure. The peak of 2960 cm−1 can be assigned to the our previous study related to the use of the lumen of HNTs for stretching vibration for −CH−, −CH −, or −CH drug the interlayer space of LAP nanodisks was structure. In the spectrum of LAP and LAP/AMX nanodisks used to encapsulate a model drug AMX. LAP nanodisks have a (Figure the moderate peak at 1640 cm−1 may be caused by two-dimensional structure with six octahedral magnesium ions the moisture from the atmosphere. The strong peak located at sandwiched between two layers of four tetrahedral silicon 1012 cm−1 can be assigned to the −Si−O− stretching vibration atoms,and the interlayer space of LAP nanodisks has been of LAP naondisks,and the broad peak at 3440 cm−1 may be proven to be used as a reservoir for drug encapsulation.
due to the bending vibration of −OH in the LAP structure. By The loading amount and the loading eﬃciency of AMX comparing the spectrum of LAP with that of LAP/AMX, a new within LAP were determined using the standard concen- peak emerged at 1770 cm−1 in the spectrum of LAP/AMX tration−absorbance (at 230 nm) calibration curve of AMX in suggesting the successful encapsulation of AMX into LAP. Due water and was optimized by changing the concentration of LAP to the quite low amount of AMX encapsulated within the LAP, and AMX, respectively. As shown in Figure an optimized it is diﬃcult to observe some other distinctive peaks of AMXdrug.
The morphology of LAP nanodisks before and after encapsulation of AMX was observed with FESEM (Figure S1,It is clear that the disk-shaped LAPdoes not signiﬁcantly change after the encapsulation of AMX,indicating the successful intercalation of drug molecules withinthe LAP interlayer space. The somewhat aggregated particlesshown in the FESEM images for both samples are presumablydue to the sample preparation method, which includes the air-drying process. As reported in our previous study, the air-dryingof the aqueous suspension of the samples before measurementmay lead to a partial aggregation or interconnection of the Figure 1. AMX loading eﬃciency as a function of AMX concentration The LAP nanodisks are able to form a stable colloidal layered under diﬀerent LAP concentrations.
structure in aqueous solution, which facilitates drug encapsu-lation.The encapsulation of drug within the LAP interlayer loading eﬃciency of 9.76 ± 0.57% could be achieved when the space may result in a change in the interlayer distance,and AMX and LAP concentration was 2 and 3 mg/mL, respectively.
this can be determined by XRD technology. The XRD patterns It is worthwhile to note that the loading eﬃciency decreases of the LAP nanodisks before and after AMX encapsulation were with the LAP concentration, which is likely due to the prone compared, and the data are shown in Figure and Table Figure 2. FTIR spectra of pure AMX (a) and LAP before (Curve 1) and after (Curve 2) AMX loading (b).
dx.doi.org/10.1021/am302130b ACS Appl. Mater. Interfaces 2012, 4, 6393−6401
ACS Applied Materials & Interfaces Figure 3. XRD patterns of LAP nanodisks before (Curve 1) and after(Curve 2) AMX loading.
Table 1. Diﬀraction Angle and Plane Spacing Data of LAPand LAP/AMX from XRD Analysis 2θ peak position (o) plane spacing (d, Å) diﬀraction plane (hkl) Obviously, most of the diﬀraction planes at their correspondingdiﬀraction angles do not change, suggesting that LAP is able tomaintain its crystalline structure after AMX The diﬀraction angle of (001) plane shifted from 6.06° to5.50°, and the plane spacing was larger (from 14.76 to 16.26 Å)after AMX encapsulation. This is likely due to the fact that theAMX molecules are intercalated along the 001 plane. Besides,when compared with AMX powder (Figure S2, no diﬀraction peaks of AMX can be detected inLAP/AMX nanodisks, which is presumably ascribed to the factthat the amount of the incorporated drug is too small to bedetectable by the XRD technique. The XRD data suggested Figure 4. SEM micrographs and diameter distribution histograms of that the incorporation of AMX within LAP is primarily via the (a) PLGA, (b) PLGA/LAP (5 wt % LAP relative to PLGA), (c) drug intercalation within the LAP interlayer space. It is also PLGA/AMX (0.5 wt % AMX relative to PLGA), and (d) PLGA/ possible that a small portion of AMX can be adsorbed onto the AMX/LAP (5 wt % LAP relative to PLGA) nanoﬁbers.
LAP surface via hydrogen bonding or other weak forces.
Formation of PLGA/LAP/AMX Nanoﬁbers. The AMX- (929 nm, Figure presumably due to the increase of the loaded LAP was then incorporated within PLGA nanoﬁbers solution conductivity, which was caused by the introduction of (with 0.5% AMX relative to PLGA) via electrospinning to form an anionic or a cationic species in the electrospinning solution.
PLGA/LAP/AMX nanoﬁbers (Scheme As controls, PLGA, Release of AMX from PLGA/LAP/AMX Composite PLGA/LAP (with 5% LAP relative to PLGA), and PLGA/ Nanoﬁbers. The in vitro drug release property of PLGA/ AMX (0.5% AMX relative to PLGA) nanoﬁbers were prepared LAP/AMX composite nanoﬁbers was investigated by exposure in the same manner. The successful incorporation of LAP of the ﬁbrous mats in PBS solution (pH = 7.4) at 37 °C. The within PLGA nanoﬁbers has been conﬁrmed by thermogravi- AMX release proﬁle was compared with those from LAP/AMX metric analysis, transmission electron microscopy, porosity nanodisks and PLGA/AMX nanoﬁbers with similar drug measurement, mechanical testing, and contact angle measure- content (Figure It is clear that the AMX in the LAP/ ment (see also Figure S3, in our AMX nanodisks has a burst release proﬁle and about 97% of the previous work.Here, in this study, SEM was used to encapsulated AMX can be released from LAP within 3 h. In characterize the morphology of the formed electrospun sharp contrast, the AMX release rate from PLGA/AMX and nanoﬁbers with diﬀerent compositions (Figure Similar to PLGA/LAP/AMX nanoﬁbers was signiﬁcantly reduced and our previous studies related to the formation of PLGA/HNTs showed a sustained manner. The release of AMX from PLGA/ composite nanoﬁbers,we were able to form electrospun AMX nanoﬁbers showed a moderate rate on the ﬁrst day, and PLGA/LAP composite nanoﬁbers with a smooth and uniform 31.8% of the AMX was released. Then, the release speed was ﬁbrous morphology even after AMX encapsulation, similar to slowed down, and approximately 100% drug release was the pure PLGA and PLGA/drug nanoﬁbers. The diameters of achieved on the ninth day. The slower AMX release rate from PLGA/LAP (550 nm, Figure PLGA/AMX (842 nm, PLGA/AMX nanoﬁbers than that from LAP/AMX nanodisks is Figure and PLGA/LAP/AMX (591 nm, Figure likely due to the eﬀective hydrogen bonding and electrostatic nanoﬁbers are smaller than that of pure PLGA nanoﬁbers interactions between the hydroxyl, amine, and carboxyl groups dx.doi.org/10.1021/am302130b ACS Appl. Mater. Interfaces 2012, 4, 6393−6401
ACS Applied Materials & Interfaces Figure 5. In vitro release of AMX from LAP/AMX nanodisks, PLGA/ Figure 6. Growth inhibition of S. aureus after treatment with AMX AMX nanoﬁbers, and PLGA/LAP/AMX nanoﬁbers.
powder (1), LAP/AMX (2), PLGA/AMX nanoﬁbers (3), and PLGA/LAP/AMX nanoﬁbers (4) with diﬀerent AMX concentrations for 24 hat 37 °C in liquid medium.
of AMX and the carboxyl residues of PLGA polymer. The drugrelease proﬁle of the PLGA/LAP/AMX composite nanoﬁbersfollows a biphasic pattern characterized by an initial fast release antibacterial activity of LAP/AMX nanodisks decreased with and a followed sustained release phase after 12 h. 40.2% of the the drug concentration. This is likely due to the increased AMX was released within the ﬁrst 12 h, and a sustained release concentration of LAP in the liquid medium, which can absorb with a relatively low rate remained; 63.5% AMX was released AMX molecules back to compromise the drug eﬃcacy to some on the 14th day. Since PLGA is biodegradable and LAP has a extent. The bacterial inhibition of PLGA/AMX and PLGA/ swelling ability in PBS solution,it is believed that all of the LAP/AMX nanoﬁbers increased with the drug concentration encapsulated AMX can be released with time. The release rate and was higher than 90% at each concentration. There was no of the PLGA/LAP/AMX nanoﬁbers was faster than that of the statistically signiﬁcant diﬀerence between pure AMX powder PLGA/AMX nanoﬁbers in the ﬁrst 2 days and then showed a and PLGA/LAP/AMX nanoﬁbers at the same AMX concen- slower and sustained release rate.
tration in terms of the bacterial inhibition eﬃcacy (p > 0.05), The burst release of the LAP/AMX nanodisks may be due to suggesting that the PLGA/LAP/AMX composite nanoﬁbers the swelling behavior of the colloidal LAP. After contact with have a comparable bacterial inhibition eﬃcacy with that of the the PBS solution, the LAP nanodisks swell and the pure AMX powder. In contrast, PLGA and PLGA/LAP incorporated AMX molecules can be quickly released. The nanoﬁbers without AMX encapsulation did not have any initial fast release of the PLGA/LAP/AMX nanoﬁbers may be antibacterial eﬃcacy, similar to the untreated negative control.
due to the inevitable release of AMX from LAP/AMX The correlation of the antibacterial activity of PLGA/LAP/ nanodisks when they were mixed with PLGA solution before AMX nanoﬁbers as a function of AMX release time was also electrospinning. After the formation of PLGA/LAP/AMX investigated (Figure S4, Apparently, nanoﬁbers, the partially released AMX can be attached onto at all the release time points (1, 2, 6, 12, 24, 48, 72, and 96 h, the nanoﬁber surface or dispersed throughout the polymer respectively), the released AMX from PLGA/LAP/AMX matrix in a matrix-type structure, thereby causing an initial burst nanoﬁbers can eﬀectively inhibit the bacterial growth, similar release. It is interesting to note that the initial burst release of to that from PLGA/AMX nanoﬁbers.
PLGA/LAP/AMX nanoﬁbers is higher than that of PLGA/ The bacterial inhibition activity of the PLGA/LAP/AMX AMX. This can be explained as follows: The diameter of composite nanoﬁbers was also tested onto solid medium.
PLGA/LAP/AMX nanoﬁbers is apparently smaller than that of Figure shows the digital photos of the antibacterial circles on PLGA/AMX (as shown in Figure which shortened the drug agar plates at diﬀerent culture times. PLGA (1), PLGA/LAP diﬀusion distance between the PLGA ﬁber matrix to the release (2), PLGA/AMX (3), and PLGA/LAP/AMX (4) nanoﬁbers medium. The followed slow release speed of the PLGA/LAP/ were pasted onto the agar plate for bacteria inhibition (Figure AMX relative to the PLGA/AMX nanoﬁbers is easily −c). Obviously, both the PLGA/AMX and PLGA/LAP/ understandable due to the coexistence of two types of drug- AMX nanoﬁbers were able to eﬀectively inhibit bacterial carriers, namely, reservoir-type and matrix-type (Scheme growth, and the zones of inhibition for PLGA/LAP/AMX and The drug should ﬁrst come out from the reservoir of LAP and PLGA/AMX are basically similar in size after a 12, 24, and 48 h then from the polymer matrix, which provides an additional culture, implying that the PLGA/LAP/AMX nanoﬁbers has a barrier for the drug good bacterial inhibition eﬃcacy under the studied conditions.
In Vitro Antibacterial Activity Assay of Nanoﬁbers. For The bacterial inhibition eﬃcacy of the PLGA/LAP/AMX development of novel, eﬀective drug delivery systems, it is nanoﬁbers was further conﬁrmed by removing the nanoﬁbrous important to maintain the activity of the drug after mats from the agar plate after a 4 h release of AMX, followed by encapsulation within the composite PLGA/LAP nanoﬁbers.
bacterial seeding (Figure −f). Similar to the above method, We next explored the in vitro antibacterial activity of the AMX- PLGA/AMX and PLGA/LAP/AMX nanoﬁbers were able to loaded nanoﬁbers using S. aureus as a model bacterium both in eﬀectively inhibit the bacterial growth. In contrast, PLGA and liquid and on solid medium. Figure shows the bacterial PLGA/LAP nanoﬁbers without AMX encapsulation did not inhibition assay results of AMX powder, LAP/AMX nanodisks, inhibit the bacterial growth in both cases, implying that the PLGA/AMX nanoﬁbers, and PLGA/LAP/AMX nanoﬁbers in bacterial inhibition eﬀect is solely related to the encapsulated liquid medium with diﬀerent AMX concentrations (10, 20, and AMX drug. It should be noted that, for solid medium testing, 30 μg/mL, respectively). The AMX powder was able to inhibit we just tested all the nanoﬁbrous samples because it was the bacterial growth at each studied concentration, while the diﬃcult to uniformly lay down the solid powder samples of free dx.doi.org/10.1021/am302130b ACS Appl. Mater. Interfaces 2012, 4, 6393−6401
ACS Applied Materials & Interfaces Figure 7. Inhibition of S. aureus cultured on agar plate incubated at 37 °C at 12, 24, and 48 h. In panels a−c, nanoﬁbers were pasted onto the agarplate for the whole culture time period. In panels d−f, nanoﬁbers were removed after a 4 h release of AMX onto the agar plate. 1−4 representsPLGA, PLGA/LAP, PLGA/AMX, and PLGA/LAP/AMX nanoﬁbers, respectively.
AMX and LAP/AMX for eﬀective comparison. Taken togetherwith the data obtained in liquid medium, we can conclude thatthe developed PLGA/LAP/AMX composite nanoﬁbers are ableto inhibit the growth of a model bacterium, and the loading ofAMX within the composite nanoﬁbers does not compromisethe inherent antibacterial activity of the drug. It should benoted that, although the PLGA/LAP/AMX and PLGA/AMXnanoﬁbers showed diﬀerent drug release patterns, theantimicrobial activity was almost the same. However, themajor advantage of the PLGA/LAP/AMX nanoﬁbers is that thesustained release of the drug from the ﬁbers is very importantfor certain biomedical applications requiring the drug to have a Figure 8. MTT viability assay of PIEC cells seeded on the TCPs long-term therapeutic eﬃcacy. Besides, the incorporation of (control), cover slips (control), PLGA nanoﬁbers, and PLGA/LAP/ LAP within PLGA nanoﬁbers can signiﬁcantly enhance the AMX nanoﬁbers (mean ± SD, n = 3, *p < 0.05).
mechanical property of PLGA nanoﬁbers.
Cytocompatibility of the PLGA/LAP/AMX Composite the time point of 8 h and 3 days does not impact the cell Nanoﬁbers. To further validate the potential biomedical applications of the developed PLGA/LAP/AMX composite The comparison of the cytocompatibility of PLGA/LAP/ nanoﬁbers, we next tested the cytocompatibility of the ﬁbers via AMX composite nanoﬁbers with pure PLGA nanoﬁbers was MTT assay in comparison with pure PLGA nanoﬁbers with also validated via the cell morphology observation. The proven biocompatibility.The viability of PIEC cells morphologies of PIEC cells cultured onto PLGA and PLGA/ cultured onto both PLGA and PLGA/LAP/AMX nanoﬁbers LAP/AMX nanoﬁbers after an 8 h and 3 day culture are shownin Figure Obviously, cells are able to attach onto both after 8 h and 3 days is shown in Figure No statistically nanoﬁbrous scaﬀolds after an 8 h culture, and after 3 days, the signiﬁcant diﬀerence can be found among each sample after an cells cultured onto both nanoﬁbrous scaﬀolds display a 8 h culture, indicating that both PLGA and PLGA/LAP/AMX phenotypic shape, indicating that the cells can penetrate and nanoﬁbers display similar adhesion viability, in comparison with migrate within the scaﬀolds in a manner similar to native the coverslips and TCPs. On day 3, the proliferation viability of extracellular matrix. These cell morphology observation data PIEC cells cultured onto both PLGA and PLGA/LAP/AMX corroborate the results of the MTT assay.
nanoﬁbers is signiﬁcantly higher than those onto coverslips andTCPs (p < 0.05), and no signiﬁcant diﬀerence exists between the PLGA and PLGA/LAP/AMX nanoﬁbers (p > 0.05). This In summary, we developed a facile approach to encapsulating implies that both PLGA and PLGA/LAP/AMX nanoﬁbers have an antibiotic drug AMX within PLGA/LAP composite an excellent cytocompatibility, and the incorporation of LAP/ nanoﬁbers for biomedical applications. The AMX-loaded LAP AMX nanodisks does not compromise the cytocompatibility of nanodisks with an optimized loading eﬃciency of 9.76 ± 0.57% PLGA nanoﬁbers. This also indicates that the released AMX at were able to be incorporated within PLGA nanoﬁbers without dx.doi.org/10.1021/am302130b ACS Appl. Mater. Interfaces 2012, 4, 6393−6401 ACS Applied Materials & Interfaces Figure 9. SEM images of PIEC cells cultured onto PLGA (a, c) and PLGA/LAP/AMX (b, d) nanoﬁbers after an 8 h (a, b) and 3 day (c, d) culture.
signiﬁcantly changing the PLGA ﬁbrous morphology. With the Donghua University Doctorate Dissertation of Excellence coexistence of both the reservoir-type of LAP interlayer space (BC201107). M.Z. thanks the National Natural Science and the matrix-type of PLGA nanoﬁbers, the release proﬁle of Foundation of China (50925312) for support.
AMX was able to be signiﬁcantly improved with a biphasic andsustained manner. Furthermore, PLGA/LAP/AMX nanoﬁbers display eﬀective antibacterial activity and noncompromisedcytocompatibility in comparison with pure PLGA nanoﬁbers.
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Rapid detection of genetically modified organisms on a continuous-flow polymerase chain reaction microfluidics
Analytical Biochemistry 385 (2009) 42–49 Contents lists available at Analytical Biochemistry Rapid detection of genetically modiﬁed organisms on a continuous-ﬂowpolymerase chain reaction microﬂuidics Yuyuan Li, Da Xing *, Chunsun Zhang MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, No. 55, Zhongshan Avenue West, Tianhe District,Guangzhou 510631, People's Republic of China
j. innov.dev.strategy. 2(2): 22-27 (July 2008) ANTIMICROBIAL SUSCEPTIBILITY OF Salmonella SEROVARS ISOLATED FROM BLOOD M. J. ISLAM1, K. K. DAS2, N. SHARMIN3, M. N. HASAN4 AND A. K. AZAD5 1Lecturer, Department of Pharmacy, University of Development Alternative, Dhanmondi, Dhaka, 2, 3 & 4Lecturer, Department of Biotechnology and Genetic Engineering, 5Assistant professor, Department of Pharmacy, University of Development Alternative, Dhanmondi, Dhaka, Bangladesh.